The invention relates to compositions and methods for producing polyhydroxyalkanoates in transformed plants and transformed host cells.
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates which are naturally produced by a large variety of bacteria and fingi. PHAs are biodegradable and renewable, thereby providing an attractive alternative to petroleum-based plastics. However, high production cost has limited the widespread use of PHAs derived from bacterial fermentation. One alternative to reduce cost, production of PHAs in agricultural crops, has been regarded as promising. Small amounts of the PHA have been produced in the cytosol, plastids and peroxisomes of genetically engineered plants. See Poirier (1999) Curr. Opin. Biotechnol. 10(2):181-5; Madison et al. (1999) Microbiol. Mol. Biol. Rev. 63(1):21-53).
PHA synthases catalyze polymerization of hydroxyacyl-CoA substrates into PHA. The substrate specificity of this class of enzymes varies across the spectrum of PHA-producing organisms. The variation in substrate specificity of PHA synthases is supported by indirect evidence observed in heterologous expression studies (Lee et al. (1995) Appl. Microbiol. Biotechnol. 42:901 and Timnm et al. (1990) Appl. Microbiol. Biotech. 33:296).
Until recently, the only PHA that has been produced in plants was polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyric acid (John et al (1996) Proc. Natl. Acad. Sci. USA 93:12768-12773; Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764; Padgette et al. (1997) Plant Physiol. 114 (Suppl.) 3S; Poirier et al. (1992) Science 256:520-523)). Because this polymer is crystalline and brittle with a melting point too close to its degradation point, PHB is difficult to mold into desirable products (Lee (1996) Biotechnol. Bioeng. 491:1-14).
Many bacteria make copolymers of 3-hydroxyalkanoic acids with a carbon chain length greater than or equal to five (Steinbuchel (1991) Biomaterials: Novel Materials from Biological Materials, ed. Byrom (New York: Macmillan Publishers Ltd.), pp. 123-213). Such copolymers are polyesters composed of different 3-hydroxyalkanoic acid monomers. Depending on the composition, these copolymers can have properties ranging from firm to elastic (Anderson et al. (1990) Microbiol. Rev. 54:450-472; Lee, (1996) Biotechnol. Bioeng. 49:1-14). Unlike the homopolymeric PHB, the PHA copolymers are suitable for a variety of applications because these copolymers exhibit a wide range of physical properties.
Initial attempts at producing PHA in plants involved producing PHA in the cytosol, but production of PHA in this cellular compartment proved toxic to the plant (Poirier et al. (1992) Science 256:520-523). This problem was overcome by targeting the PHA-producing enzymes to plastids (Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764). In either cellular compartment, however, only PHB was accumulated, not any of the copolymers. With both of these methods, the genes from Ralstonia eutropha were used. The PHA synthase of this bacterium can utilize only short chain (C3-C5) monomers (Steinbuchel (1991) Biomaterials: Novel Materials from Biological Materials, ed. Byrom (New York: Macmillan Publishers Ltd.), pp. 123-213). Later, copolymer production in Arabidopsis and canola was reported by Slater et al. (1999) Nature Biotechnology 17: 1011-1016.
The synthesis of PHA containing 3-hydroxyalkanoic acid monomers ranging from six to sixteen carbons in Arabidopsis thaliana was reported (Mittendorf et al. (1998) Proc. Natl. Acad. Sci. USA 95:13397-13402). To accumulate PHA, the Arabidopsis plants were transformed with a nucleotide sequence encoding PHA synthase from Pseudomonas aeruginosa that was modified for peroxisome targeting by the addition of a nucleotide sequence encoding the C-terminal 34 amino acids of a Brassica napus isocitrate lyase. In these plants, PHA was produced in glyoxysomes, leaf-type peroxisomes, and vacuoles. However, PHA production was very low in the Arabidopsis plants, suggesting that either the introduced PHA synthase did not function properly in the intended organelle, or more likely that the necessary substrates for the introduced PHA synthase were present at levels that were limiting for PHA synthesis. While this report demonstrated that PHA can be produced in peroxisomes of plants, the level of PHA produced in the plants was far below levels necessary for the commercial production of PHA in plants. Thus, methods and compositions directed to increasing the level of substrate for PHA synthases are needed for production of PHA in plants.
There are two types of fatty acid synthase (FAS). In type I FAS, various enzyme activities are located on different domains of a multifunctional protein. In type II FAS, these enzyme activities are catalyzed by individual polypeptides. 3-oxoacyl-[acyl carrier protein(ACP)] reductase (OAR) is a component of the type II FAS. This enzyme reversibly reduces xcex2-ketoacyl-ACP, the condensation product of an acetyl residue and a nascent acyl-ACP, to xcex2-hydroxyacyl-ACP. In vitro, OAR also uses 3-ketoacyl-CoA as a substrate to catalyze formation of 3-hydroxyacyl-CoA. This use of 3-ketoacyl-CoA is at a lower efficiency than the use of xcex2-ketoacyl-ACP as substrate (Shimakata et al. (1982) Arch. Biochem. Biophys. 218(1): 77-91).
NADPH-dependent OAR from Spinacia oleracea has been described to catalyze the forward reaction of reducing xcex2-ketoacyl-ACP, more than seventeen times faster than the reverse dehydrogenation reaction, at neutral or acidic pH. This OAR has also been shown to use only D-3-hydroxybutyryl-ACP as a substrate but not the L-form counterpart.
NADH-dependent forms of OARs have been described from plant species such as castor bean and avocado (Shimakata et al. (1982) Arch. Biochem. Biophys. 218(1): 77-91; Caughey et al. (1982) Eur. J. Biochem. 123(3): 553-61). Taguchi et al. have shown that over-expression of a bacterial NADPH-dependent OAR increases D-3-hydroxyacyl-CoA monomer supply for PHA synthase and leads to accumulation of PHAs in E. coli (Taguchi et al. (1999) Fems. Microbiol. Lett. 76(1): 183-190).
Thus, methods and compositions directed to plant OARs are needed for increasing the level of substrate for PHA synthases, and for production of PHA in plants.
Compositions and methods directed to producing PHA in host cells and plants are provided, including PHA copolymers. The compositions are directed to isolated nucleic acid molecules encoding 3-oxoacyl-[acyl carrier protein(ACP)] reductase (OAR) polypeptides. Expression cassettes comprising the nucleotide sequences encoding the OAR enzymes are also provided.
For PHA production in host cells, such as bacteria, with one or more endogenous PHA synthases, the methods involve genetically manipulating the host cell to produce one or more OAR enzymes. The methods comprise stably integrating in the genome of a host cell nucleotide sequences encoding OAR enzymes.
For PHA production in plants, the methods involve genetically manipulating a plant to produce one or more OAR enzymes. If desired, the plants can also be transformed with nucleotide sequences encoding additional enzymes that are necessary for, or favorably affect, the synthesis of PHA in the plants. Such enzymes include, for example, one or more PHA synthases. The OAR enzymes, and any other desired enzymes, can be targeted in the plant to the peroxisomes by operably linking a peroxisome-targeting sequence to a sequence encoding the enzyme. The methods comprise stably integrating in the genome of a plant nucleotide constructs comprising nucleotide sequences encoding OAR enzymes, PHA synthases, and/or any other desired enzymes for PHA synthesis in a plant or part thereof.
Also provided are plants, plant tissues, plant cells, and seeds thereof, that are genetically manipulated to produce one or more OAR enzymes. Further provided are plants, plant tissues, plant cells, and seeds thereof comprising stably integrated in their genomes a nucleotide sequence encoding an OAR and a nucleotide sequence encoding a PHA synthase. Such plants, plant tissues, plant cells, and seeds can additionally comprise stably integrated in their genomes one or more additional nucleotide sequences which encode enzymes that favorably affect PHA synthesis.